CN103087935A - Fermentation method of conjugated linoleic acid and strain used in method - Google Patents
Fermentation method of conjugated linoleic acid and strain used in method Download PDFInfo
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- CN103087935A CN103087935A CN2013100151505A CN201310015150A CN103087935A CN 103087935 A CN103087935 A CN 103087935A CN 2013100151505 A CN2013100151505 A CN 2013100151505A CN 201310015150 A CN201310015150 A CN 201310015150A CN 103087935 A CN103087935 A CN 103087935A
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- linoleic acid
- linoleate isomerase
- saccharomyces cerevisiae
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Abstract
The invention discloses a fermentation method of conjugated linoleic acid and a strain used in method, and relates to a method in which linoleate isomerase is expressed by using a saccharomyces cerevisiae surface display system, and capable of catalytic synthesis of t10, c12-conjugated linoleic acid by using the linoleate isomerase. An expression vector containing a propionibacterium acnes derived linoleate isomerase gene (pai) is constructed and a saccharomyces cerevisiae strain containing the expression vector is obtained. After the induction culture, the linoleate isomerase is displayed and expressed in saccharomyces cerevisiae, and the linoleate isomerase is added into a buffer solution containing substrate linoleic acid for catalytic reaction to generate the t10, c12-conjugated linoleic acid. According to the method provided by the invention, the linoleate isomerase is linked to cytoderm of the saccharomyces cerevisiae by the microorganism surface display system, so that extracellular substrates and the linoleate isomerase can be fully contacted, the catalysis efficiency is improved, a single isomer with physiological activity can be obtained, separation and purification steps are simplified and the production cost is reduced.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to structure and the fermentation of bacterial strain, especially a kind of fermentation process of conjugated linolic acid and bacterial strain uses therefor.
Background technology
Conjugated linolic acid (Conjugated linoleic acid, be called for short CLA) be the octadecadienoic acid with conjugated double bond, the derivative a series of positions that form of lipid acid linolic acid (linoleic acid is called for short LA) of needed by human and the general name of conformer.That CLA has is anticancer, atherosclerosis, reduction cholesterol, regulate blood sugar, enhancing body immunological competence, promote the multiple physiologically actives such as growth, and wherein, t10, c12-CLA are one of isomer of tool physiologically active.
At occurring in nature, CLA extensively is present in the meat product and milk-product of ruminating animal, but content is extremely low.At present, industrial production CLA mainly adopts the chemical isomerization method, namely by linoleic alkali isomerization.But the product that this method obtains is the mixture of multiple isomer, except the isomer with physiologically active, also contain the by product of multiple security the unknown, and the separation and purification of active isomer is very difficult.Adopt genetic engineering technique, acquisition can be expressed the bacterial strain of linoleate isomerase, utilize the linoleate isomerase of self that LA is changed into CLA, mild condition, and can synthesize the individual isomer with physiologically active, not only can greatly simplify the step of later separation purifying, reduce production costs, and for future CLA be used for the rational proportion that clinical trial and meals add different CLA isomer, have advantages of that all mixed isomers is incomparable.The linoleate isomerase (pai) that derives from propionibacterium acnes (Propionibacterium acnes) is the linoleate isomerase of unique a kind of known crystalline structure, LA can be changed into single active isomer t10, c12-CLA, and successful expressing in the multiple systems such as intestinal bacteria, yeast, tobacco and rice.
Summary of the invention
The object of the invention is to utilize the yeast saccharomyces cerevisiae surface display system to express the linoleate isomerase that derives from propionibacterium acnes, successfully built a strain by showing that linoleate isomerase can catalyze and synthesize single-activity isomer t10, the Yeast engineering bacterium strain of c12-CLA, and utilize this bacterial strain synthetic C t10,c12-onjugated linoleic acid that successfully ferments.
The present invention realizes that the technical scheme of purpose is as follows:
A kind of genetic engineering bacterium of expressing Recombinant Express of Conjugated Linoleic Acid Isomerase Gene at cell walls contains linoleate isomerase gene, and gene order is seen sequence 3, and the pYD1 of expression vector, Host Strains are yeast saccharomyces cerevisiae EBY100.
And described conjugated linolic acid is C t10,c12-onjugated linoleic acid.
Express the cultural method of the genetic engineering bacterium of Recombinant Express of Conjugated Linoleic Acid Isomerase Gene at cell walls, step is: described genetic engineering bacterium is inoculated in the YNB-CAA substratum that contains semi-lactosi, and inducing culture, centrifugal, collect thalline.
And described galactose concentration is 1-2% (W/V).
And described inducing culture temperature is 20-25 ° of C, and the inducing culture time is 48-96h.
And, described engineering bacteria is joined to contain in the linoleic damping fluid of substrate carry out oscillatory reaction, obtain conjugated linolic acid after reaction.
And described pH of buffer is 6.5-7.5.
And, described damping fluid Sodium phosphate dibasic-citrate buffer solution.
And described oscillatory reaction temperature is 30-37 ° of C, and the oscillatory reaction time is 4-36h.
A kind of whole-cell catalyst for preparing conjugated linolic acid contains genetic engineering bacterium as claimed in claim 1, and described conjugated linolic acid is C t10,c12-onjugated linoleic acid.
Advantage of the present invention and positively effect are as follows:
1, the present invention is by in the linoleate isomerase gene insertion vector pYD1 that will derive from propionibacterium acnes (Propionibacterium acnes), built expression vector pYD1::pai, and it is imported in yeast saccharomyces cerevisiae EBY100, acquisition contains the Wine brewing yeast strain EBY100-pYD1-pai of expression vector pYD1::pai, this bacterial strain is after inducing culture, the PAI success be illustrated in the brewing yeast cell wall surface, can fully contact with the substrate linolic acid, and LA is converted into single-activity isomer t10, c12-CLA.
2, the present invention utilizes the yeast saccharomyces cerevisiae surface display system first, linoleate isomerase gene is illustrated in the cell walls surface, make substrate can with its sufficient contact, can either improve the catalytic efficiency of linoleate isomerase, can obtain to have again the individual isomer of physiologically active, do not need to be further purified again separation, enhance productivity.
3, the present invention utilizes the microorganism surface display system that linoleate isomerase is connected on the brewing yeast cell wall, the extracellular substrate is fully contacted with enzyme, improve catalytic efficiency, and can obtain to have the individual isomer of physiologically active, save purification procedures, reduce production costs.
Description of drawings
Fig. 1 is yeast saccharomyces cerevisiae surface display system principle schematic of the present invention.
Fig. 2 is the structural representation of expression vector pYD1::pai of the present invention.
Fig. 3 is that pYD1::pai transformed saccharomyces cerevisiae PCR of the present invention identifies electrophoresis result figure.
Fig. 4 is that EBY100-pYD1-pai surface display linoleate isomerase of the present invention catalyzes and synthesizes t10, the determination of yield figure of c12-CLA.
Fig. 5 is that EBY100-pYD1-pai surface display linoleate isomerase of the present invention catalyzes and synthesizes t10, the gas chromatogram of c12-CLA.
Embodiment
Below in conjunction with embodiment, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Content of the present invention comprises: with the expression plasmid of natural linoleate isomerase pai gene, what contain the PAI expression plasmid can produce t10, the Wine brewing yeast strain of c12-CLA, the construction process of described Wine brewing yeast strain and described Wine brewing yeast strain are produced t10, the method for c12-CLA.
The present invention has built expression vector pYD1::pai by in the pai gene insertion vector pYD1 that will derive from P.acnes, and it is imported in yeast saccharomyces cerevisiae EBY100, obtains to contain the Wine brewing yeast strain EBY100-pYD1-pai of expression vector pYD1::pai.Above-mentioned bacterial strains after inducing culture, the PAI success be illustrated in the brewing yeast cell wall surface, can fully contact with the substrate linolic acid, and LA is converted into single-activity isomer t10, c12-CLA.
Described natural linoleate isomerase gene (pai) derives from propionibacterium acnes (Propionibacteriumacnes), and the bacterial strain deposit number is ATCC6919, and No. GenBank of this gene is AX062088.1, and its nucleotide sequence is as shown in sequence 3.
Described expression plasmid inserts initial carrier pYD1(available from invitrogen company with the pai gene, article No. V83501); Expression plasmid after described yeast saccharomyces cerevisiae will build changes (available from invitrogen company, article No. C83900) in initial strains yeast saccharomyces cerevisiae EBY100 over to;
The method of described yeast saccharomyces cerevisiae surface display linoleate isomerase is, yeast saccharomyces cerevisiae is inoculated in YNB-CAA, and is centrifugal through the semi-lactosi inducing culture, collects thalline, and washing obtains the Wine brewing yeast strain that surface display has linoleate isomerase; Described galactose concentration is 1-2%; Described inducing temperature is 20-25 ° of C; Described induction time is 48-96h;
The method that described yeast saccharomyces cerevisiae surface display linoleate isomerase catalyzes and synthesizes conjugated linolic acid is, the Wine brewing yeast strain of surface display linoleate isomerase is joined as whole-cell catalyst contain (pH6.5-7.5) in linoleic damping fluid, oscillatory reaction; Described damping fluid is SODIUM PHOSPHATE, MONOBASIC-citrate buffer solution; Described temperature of reaction is 30-37 ° of C; The described reaction times is 4-36h;
One, the structure of yeast saccharomyces cerevisiae surface display system expression vector
Take the genome of propionibacterium acnes (Propionibacterium acnes) as template, utilize the primer in table 1 to carry out pcr amplification.
The PCR reaction system is: 2 * PCR Buffer(contains Mg
2+) 25 μ l, dNTP(25mM) 5 μ l, upstream primer PAI-F and downstream primer PAI-R(10 μ M) each 1 μ l, template (propionibacterium acnes complete genome DNA) 1 μ l, KOD Fx archaeal dna polymerase 0.5 μ l, adding sterilized water to final volume is 50 μ l.
The PCR reaction conditions is: 94 ℃ of denaturation 2min, and 98 ℃ of sex change 10s, 56 ℃ of annealing 30s, 68 ℃ are extended 90s, react 35 circulations, extend 10min after 68 ℃.
Table 1 the primer sequence
Acquired goal gene pai is carried out double digestion with EcoRI and XhoI, be connected with the plasmid fragment pYD1 that processed through same restriction endonuclease after reclaiming, to connect product and transform escherichia coli jm109 competent cell, and evenly coat on LB flat board with amicillin resistance (100 μ g/ml), 37 ℃ of overnight incubation, the picking mono-clonal carries out bacterium colony PCR checking and enzyme and cuts checking, obtain expression vector pYD1::pai, see Fig. 2.
Two, the structure that contains the Wine brewing yeast strain of expression vector
Composition without amino yeast nitrogen (YNB) solid screening culture medium: glucose 2%, yeast nitrogen alkali 0.67%, ammonium sulfate 0.5%, agar 2%.
initial strains yeast saccharomyces cerevisiae EBY100 is that the Tryptophan auxotrophic mutant bacterial strain is (available from invitrogen, article No. C83900, Tryptophan auxotrophic mutant is to screen used for the later stage, carrier pYD1 contains the TRP1 gene, transform the synthesis capability that to recover its tryptophane after bacterial strain EBY100, just can grow on without amino yeast nitrogen (YNB) solid screening culture medium, and EBY100 can not grow on YNB, can only grow containing on the YPD substratum of tryptophane), initial carrier pYD1 contains the TRP1 gene, the Wine brewing yeast strain that contains expression vector can be grown on the substratum of tryptophane disappearance, therefore select YNB solid screening culture medium.
With above-mentioned acquired expression vector transformed saccharomyces cerevisiae EBY100, concrete grammar is:
⑴ yeast saccharomyces cerevisiae EBY100 carries out three rides on the YPD flat board, cultivate 48h, obtain single bacterium colony for 30 ℃;
⑵ single bacterium colony of picking is inoculated in the YPD liquid nutrient medium, 30 ℃ of shaking culture to 2 * 10
7Cell/ml;
⑶ 4 ° of C, 5000rpm are centrifugal, collect thalline, and the thalline Eddy diffusion in sterilized water, is repeated once;
⑷ 4 ° of C, 5000rpm are centrifugal, collect thalline, with the thalline Eddy diffusion in 50 μ l0.1M lithium acetate damping fluids;
⑸ add successively 50%PEG3350240 μ l, 1M lithium acetate 36 μ l, 2mg/ml salmon essence single stranded DNA 50 μ l(to boil 5min before using in above-mentioned competent cell, be placed in 2min on ice), plasmid to be transformed (carrier pYD1::pai) 10 μ l, as enter sterilized water to final volume 360 μ l, thermal agitation mixing;
⑹ 30 ℃ of water-bath 30min, 42 ℃ of heat-shocked 40min, 8000rpm is centrifugal, collects thalline, adds the resuspended thalline of 1mlYPD liquid nutrient medium, and 4h is cultivated in 30 ℃ of recoveries;
⑺ evenly be applied to the yeast saccharomyces cerevisiae bacteria suspension on the YNB flat board, and 30 ° of C cultivate 2-4d, obtain single bacterium colony.
5 pYD1::pai transformants of picking take PAI-F, PAI-R as primer, carry out respectively the bacterium colony PCR checking of linoleate isomerase gene to above-mentioned transformant.
The PCR reaction system is: 10 * PCR Buffer2 μ l, MgCl21.6 μ l, dNTP(10mM) 0.4 μ l, upstream primer PAI-F and downstream primer PAI-R(10 μ M) each 0.8 μ l, template 1 μ l, Taq archaeal dna polymerase 0.2 μ l, adding sterilized water to final volume is 20 μ l.
the PCR reaction conditions is: 94 ° of C denaturation 2min, 96 ℃ of sex change 40s, 56 ℃ of annealing 1min, 72 ℃ are extended 90s, react 35 circulations, 72 ° of C extend 5min again, result as shown in Figure 3, the first positive contrast of swimming lane, take plasmid pYD1::pai as the pcr amplification template, the second negative contrast of swimming lane, take the genomic dna of starting strain EBY100 as the pcr amplification template, the pcr amplification product that rear five swimming lanes carry out as template for the whole cell DNA take 5 transformants, compare with the negative control bacterial strain, positive transformant can amplify the band of pai gene (1275bp), illustrate that expression vector pYD1::pai successfully changes in yeast saccharomyces cerevisiae EBY100.
Three, contain abduction delivering and the converted product analysis of the yeast saccharomyces cerevisiae of expression vector
The composition of YNB-CAA inducing culture: semi-lactosi 2%, yeast nitrogen alkali 0.67%, ammonium sulfate 0.5%, CAA0.5%.
After above-mentioned Wine brewing yeast strain activation, be inoculated in the YNB-CAA substratum, 20 ℃ of inducing culture 48h, 6000rpm is centrifugal, collect somatic cells, with the somatic cells collected with 0.9% physiological saline washed twice, join and contain in the linoleic Sodium phosphate dibasic-citrate buffer solution of 4mg/ml, 30 ℃, 200rpm shaking culture carry out conversion reaction, measure at regular intervals t10, the output of c12-CLA, with the converted product of the brewing yeast cell that contains empty carrier pYD1 as negative control.Result as shown in Figure 4, along with the increase in reaction times, t10, the output of c12-CLA increases gradually, wherein, at initial reaction stage 0-4h, t10, c12-CLA output increases significantly, 4-20h subsequently, t10, the c12-CLA increase of production is slow, and reaches maximum at 20h.Wherein, the maximum production that EBY100-pYD1-pai generates CLA is 18.2 μ g/ml, and the yeast saccharomyces cerevisiae somatic cells that contains empty carrier pYD1 can't detect t10, the generation of c12-CLA.
Above-mentioned brewing yeast cell is transformed the product of 20h, with 2 times of volume chloroform/methanol solution (2:1, V/V), oscillation extraction is got organic phase and is revolved steaming in 30 ℃, removes organic solvent, in product after revolving steaming, add chloroform dissolving mixing, add sulfuric acid/methanol solution (4%, V/V), heating in water bath for reaction 1h in 75 ℃ makes its abundant esterification, reaction adds chromatographically pure normal hexane, oscillation extraction after finishing, drying is filtered, and is used for gas chromatographic detection.Result as shown in Figure 5, the gas chromatogram of figure A:CLA methyl esters standard specimen, figure B: contain the gas chromatogram of the negative control bacterial strain converted product of empty carrier pYD1, figure C: the gas chromatogram that contains the Wine brewing yeast strain EBY100-pYD1-pai converted product of expression vector pYD1:pai.The gas chromatogram of CLA methyl esters standard specimen shows, two kinds of isomer have obtained reasonable separation, c9 wherein, and the retention time of t11-CLA is 19.29min, t10, the retention time of c12-CLA is 19.63min(Fig. 5 A).Contain the generation (Fig. 5 B) that there is no CLA in the converted product of negative control bacterial strain of empty carrier pYD1.Converted product to EBY100-pYD1-pai carries out gas chromatographic analysis, this bacterial strain catalysis LA transforms the product that generates and typical peaks (Fig. 5 C) occurs at retention time 19.56min, by with the comparing of CLA methyl esters standard specimen retention time, show that this product is t10, c12-CLA.
Claims (10)
1. genetic engineering bacterium of expressing Recombinant Express of Conjugated Linoleic Acid Isomerase Gene at cell walls, it is characterized in that: contain linoleate isomerase gene, gene order is seen sequence 3, the pYD1 of expression vector, Host Strains are yeast saccharomyces cerevisiae EBY100.
2. genetic engineering bacterium of expressing linoleate isomerase gene at cell walls according to claim 1, it is characterized in that: described conjugated linolic acid is C t10,c12-onjugated linoleic acid.
3. cultural method of expressing the genetic engineering bacterium of Recombinant Express of Conjugated Linoleic Acid Isomerase Gene at cell walls as claimed in claim 1, it is characterized in that: step is: described genetic engineering bacterium is inoculated in the YNB-CAA substratum that contains semi-lactosi, inducing culture, centrifugal, collect thalline.
4. method according to claim 3, it is characterized in that: described galactose concentration is 1-2% (W/V).
5. method according to claim 3, it is characterized in that: described inducing culture temperature is 20-25 ° of C, and the inducing culture time is 48-96h.
6. utilize the method for genetic engineering bacterium fermentation conjugated linolic acid as claimed in claim, it is characterized in that: described engineering bacteria is joined to contain in the linoleic damping fluid of substrate carry out oscillatory reaction, obtain conjugated linolic acid after reaction.
7. method according to claim 6, it is characterized in that: described pH of buffer is 6.5-7.5.
8. method according to claim 6, is characterized in that: described damping fluid Sodium phosphate dibasic-citrate buffer solution.
9. method according to claim 6, it is characterized in that: described oscillatory reaction temperature is 30-37 ° of C, and the oscillatory reaction time is 4-36h.
10. whole-cell catalyst for preparing conjugated linolic acid, it is characterized in that: contain genetic engineering bacterium as claimed in claim 1, described conjugated linolic acid is C t10,c12-onjugated linoleic acid.
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Cited By (2)
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| CN105154346A (en) * | 2015-06-26 | 2015-12-16 | 中国环境科学研究院 | Genetic recombination brewer's yeast for degrading protein, construction method and application |
| CN112695024A (en) * | 2019-10-23 | 2021-04-23 | 江南大学 | Linoleic acid isomerase and application thereof in conjugated linoleic acid production |
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| WO2001000846A2 (en) * | 1999-06-30 | 2001-01-04 | Dcv, Inc. D.B.A Bio-Technical Resources | Linoleate isomerase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154346A (en) * | 2015-06-26 | 2015-12-16 | 中国环境科学研究院 | Genetic recombination brewer's yeast for degrading protein, construction method and application |
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| CN112695024A (en) * | 2019-10-23 | 2021-04-23 | 江南大学 | Linoleic acid isomerase and application thereof in conjugated linoleic acid production |
| CN112695024B (en) * | 2019-10-23 | 2022-08-23 | 江南大学 | Linoleic acid isomerase and application thereof in conjugated linoleic acid production |
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Application publication date: 20130508 |